The present invention concerns compounds that selectively bind to expanded CAG repeat regions in DNA which encode expanded polyglutamine regions in certain disease proteins, methods of treatment employing such compounds, and assay procedures employing such compounds.
Eight inherited neurodegenerative diseases, including Huntington""s disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinobulbar muscular atrophy and spinocerebellar ataxia types 1, 2, 3, 6 and 7, are caused by expanded CAG repeats in the coding region of the disease genes (Koshy, B. T. and Zoghbi, H. Y., Brain Pathol 7, 927-942 (1997); Paulson, H. L. and Fischbeck, K. H, Ann Rev Neurosci, 19, 79-107 (1996); Paulson, H. L., Am J Hum Genet 64, 339-3450 (1999)). The CAG codon is translated into glutamine (Q), and the polyglutamine domain is the only region of homology among the eight disease proteins. The length of the repeat is the critical determinant of age-of-disease onset, with repeat length greater than 40 glutamines producing neurodegeneration in seven of the eight diseases.
Proteins with pathologic-length polyglutamine domains display novel properties that are not present in these proteins when they contain a shorter polyglutamine domain. Length-dependent polyglutamine-protein interactions are reported for Huntington-associated protein 1 (HAP-1), GAPDH, leucine-rich acidic nuclear protein, vimentin, neurofilament, apopain, calmodulin, WW domain proteins and Ras-related nuclear protein/ARA24 (Li, X. J. et al., Nature 378, 398-402 (1995); Burke, J. R. et al, Nature Med 2, 347-350 (1996); Matilla, A. et al. Nature 389, 974-978 (1997); Onodera, 0. et al., Biochem and Biophys Res Commun 238, 599-605.(1997); Nagai, Y. et al. m Exp Neurol 155, 195-203 (1999); Goldberg, Y. P. et al., Nature Genet 13, 442-449 (1996); Bao, J. et al., Proc Natl Acad Sci USA 93, 5037-5042 (1996); Faber, P. W. et al., Hum Mol Genet 7, 1463-1474. (1998); Hsiao, P. W. et al., J Biol Chem 274, 20229-20234 (1999)).
Proteins with expanded polyglutamine domains also aggregate and aggregation is a pathologic hallmark of the polyglutamine repeat diseases (Hackam, A. S. et al. J Cell Biol 141, 1097-1105 (1998); Perez, M. K. et al. J Cell Biol 143, 1457-1470 (1998)). These polyglutamine length-dependent properties may arise from the ability of long polyglutamine domains to adopt unique three-dimensional conformations and serve to confer the disease proteins with a pathologic gain-of-function (Perutz, M. F. Trends Biochem Sci 24, 58-63 (1999); Lansbury, P. T. J. Proc Natl Acad Sci USA 96, 3342-3344 (1999)).
Lansbury proposed that during the initial stages of folding of expanded-polyglutamine proteins, misfolded intermediates interact with themselves (homologous interactions) or other proteins (heterologous interactions) leading to critical cell injury (supra). Supporting this hypothesis of length-dependent alteration in tertiary structure, Trottier et al. identified a monoclonal antibody (mAb1C2) that preferentially recognizes proteins with long, but not short, polyglutamine domains (Trottier, Y. et al., Nature 378, 403-406 (1995)). Shorter peptides that selectively bind pathologic-length polyglutamine regions would be useful in inhibiting interaction with other proteins, thereby slowing, or preventing, disease pathology, and in rational drug design. However, such peptides have not previously been developed.
Accordingly, the present invention provides compounds that selectively bind to expanded polyglutamine repeats. For example, such a compound is characterized in that it binds to a first polyglutamine peptide consisting of 60 glutamine residues under conditions in which the compound does not bind to a second polyglutamine peptide consisting of 20 glutamine residues. The compound are represented by Formula I:
xe2x80x83X1xe2x80x94R11R12R13R14xe2x80x94Y1xe2x80x83xe2x80x83(1)
wherein:
R11 is Trp;
R12 is (i) Trp or (ii) a charged amino acid such as Lys, Arg or His (preferably Lys or Arg, and most preferably Lys);
R13 is (i) Trp or (ii) a charged amino acid such as Lys, Arg or His (preferably Lys or Arg, and most preferably Lys);
subject to the proviso that one of R12 and R13 is Trp and the other is a charged amino acid;
R14 is Trp;
X1 is a polypeptide consisting of from zero to 5, 10 or 20 or 30 amino acids, preferably standard amino acids; and
Y1 is a polypeptide consisting of from zero to 5, 10 or 20 or 30 amino acids, preferably standard amino acids;
or a physiologically or pharmaceutically acceptable salt thereof. Compositions comprising compounds as described above in a pharmaceutically acceptable carrier, and the use of such compounds for the preparation of a medicament for the treatment of disorders as described herein, are also aspects of the present invention.
Compounds of the invention may be conjugated to a detectable group (e.g., a fluorescent group, an enzyme), a heterologous protein or peptide, etc. in accordance with known techniques. Detectable groups may or may not be a heterologous protein or peptide. The heterologous protein or peptide may be a translocation peptide (or xe2x80x9ctransduction peptidexe2x80x9d).
A second aspect of invention is a nucleic acid (e.g., a DNA, an RNA) encoding a compound as described above (including those conjugated to a heterologous protein or peptide), along with constructs comprising such nucleic acids operatively associated with a promoter, transfer vectors (e.g., plasmids, viruses, etc.) containing such constructs, and cells that contain and express such nucleic acids and constructs.
A third aspect of the present invention is a method of treating a cell that contains and expresses a protein having an expanded polyglutamine region, the method comprising administering to the cell a treatment effective amount of a compound as described above. The administering step may be carried out directly, e.g., by administering the compound per se (including as a pharmaceutically acceptable salt) directly to the cell, or indirectly by administering a vector that encodes and expresses the compound in the cell (with the nucleic acid that encodes and expresses that compound acting as an intermediate). The compound may be administered as a conjugate with a heterologous protein or peptide, particularly a translocation peptide, as described above.
A fourth aspect of the invention is a method of treating a subject afflicted with a nneurodegenerative disease characterized by the presence of expanded polyglutamine repeats. The method comprises administering to the subject a treatment effective amount of a compound as described above. Again, the administering step may be carried out directly, by administering the compound per se, or indirectly by administering a vector that encodes and expresses the compound in cells of the subject being treated. Again, the compound may be administered as a conjugate with a heterologous protein or peptide, particularly a translocation peptide, as described above.
A further aspect of the invention is the use of compounds or vectors as described above for the preparation of a medicament for carrying out a method as described above.
A fifth aspect of the invention is a method of detecting an expanded polyglutamine domain in a sample suspected of containing the same. The method comprises the steps of: (a) contacting a sample suspected of containing an expanded polyglutamine domain to a compound as described above, and then (b) detecting the presence or absence of binding of the compound to the sample, the presence of binding indicating the presence of an expanded polyglutamine domain in the sample. typically, the method is carried out by conjugating the compound to a detectable group, and then determining the presence or absence of binding of the detectable group to the sample. For example, the compound may be conjugated to thioredoxin, and the detecting step may be carried out by turbidometric assay.
A sixth aspect of the present invention is a method of screening compounds (e.g., in a high throughput screening procedure) for activity in treating an expanded polyglutamine repeat disease. The method comprises the steps of (a) providing a reagent system comprising an expanded polyglutamine segment conjugated to thioredoxin; (b) combining the test compound with the reagent system; and then (c) determining the presence or absence of aggregation in the reagent system (e.g., by turbidometric assay) the absence of aggregation indicating that the compound is a candidate for activity in treating an expanded polyglutamine repeat disease.
A seventh aspect of the present invention is a method of screening compounds (e.g., in a high throughput screening procedure) for activity in treating an expanded polyglutamine repeat disease. The method comprises the steps of (a) providing a reagent system comprising: (i) a first compound comprising an expanded polyglutamine segment conjugated to a first signal group and (ii) a second compound comprising an expanded polyglutamine segment conjugated to a second signal group, wherein the first and second fluorescent groups when conjugated together generate a detectable event (e.g., the emission of a signal, the quenching of a signal), and wherein each of the expanded polyglutamine segments consists of at least 40 polyglutamine residues; then (b) combining the test compound with the reagent system; and then (c) determining the presence or absence of the detectable signal,; the absence of the detectable signal indicating that the compound is a candidate for activity in treating an expanded polyglutamine repeat disease. A preferred embodiment includes: (i) a first compound comprising an expanded polyglutamine segment conjugated to a first fluorescent group and (ii) a second compound comprising an expanded polyglutamine segment conjugated to a second fluorescent group, wherein the first and second fluorescent groups are members of a fluorescence resonance energy transfer (FRET) pair, and wherein each of the expanded polyglutamine segments consists of at least 40 polyglutamine residues; then (b) combining the test compound with the reagent system; and then (c) determining the presence or absence of fluorescence resonance energy transfer between the first and second fluorescent groups; the absence of fluorescence resonance energy transfer indicating that the compound is a candidate for activity in treating an expanded polyglutamine repeat disease.
The present invention is explained in greater detail in the drawings herein and the specification set forth below.